Ps-spcl searching apparatus and method using surface plasmon resonance

ABSTRACT

A Positional Scanning—Synthetic Peptide Combinatorial Library (PS-SPC) searching apparatus and method using Surface Plasmon Resonance (SPR) are provided. The method includes spotting and fixing each of a plurality of peptide pools to a top of one thin metal film, inputting specific materials to the top of the thin metal film, applying a TM-mode light to a bottom of the thin metal film and exciting SPR for the thin metal film, and detecting a TM mode reflected light reflected from the thin metal film and displaying the detected light as a two-dimensional image.

TECHNICAL FIELD

The present invention relates to an apparatus for searching a molecularreaction using

Positional Scanning -Synthetic Peptide Combinatorial Library (PS-SPCL)that is a kind of peptide library. More particularly, the presentinvention relates to a PS-SPCL searching apparatus and method usingSurface Plasmon Resonance (SPR), for displaying the PS-SPCL searchingresult as an image using SPR.

The invention has been supported by ITR&D Program of MIC/IITA[2006-S-007-02, Ubiquitous Health Monitoring Module and SystemDevelopment].

BACKGROUND ART

Peptide library refers to a combination of hundreds to thousands ofpeptides, a combination and sequence of which are arbitrarily variedusing combinatorial chemistry. The peptide library is used for thedevelopment of a potential preliminary new medicine (a lead compound)having a biological activity.

PS-SPCL is a kind of peptide library. The PS-SPCL is a combination ofmixtures that are prepared in a way of fixing one of 19 amino acids(except cysteine) in a specific position and connecting mixtures of the19 amino acids in remaining positions. The PS-SPCL is used for thedevelopment of new peptide hormone materials, the definition of antibodyepitopes, and the development of antimicrobial, antibiotics, enzymedetergent, and physiological active peptide.

FIG. 1 is a schematic diagram illustrating a scheme of PS-SPCL. As shownin FIG. 1, in mixtures of the first group of the PS-SPCL, differentspecific amino acids each are fixed in first positions and random aminoacids are placed in remaining positions at the same rate. In mixtures ofthe second group, different specific amino acids each are fixed insecond positions and random amino acids are positioned in remainingpositions at the same rate. Also, in mixtures of the third group,different specific amino acids each are fixed in third positions andrandom amino acids are placed in remaining positions. In the above, apeptide synthesis process controls positioning specific amino acids inspecific positions only and positioning random amino acids in remainingpositions.

Thus, the PC-SPCL is composed of a plurality of peptide pools in whichdifferent specific amino acid is fixed in each sequence position.

For example, if pentamer PS-SPCL is made from 20 amino acids, 20 5=100different peptide pools become one set.

The completed set is, as shown in FIG. 2, used to perform a screeningprocess suitable to each purpose such as a reaction with specificmaterials or influence on enzyme. As a result, obtained is the result asto whether the most desired effect is obtained when any amino acids arepositioned in first positions and also, whether it is most effectivethat any amino acids are positioned in second and third positions. Ingeneralization, a linear peptide sequence that is a disclosure of anoptimal amino acid by position is secured.

However, the PS-SPCL used in this process consumes a long time and manyefforts and costs in its manufacturing because of its characteristic,but the PS-SPCL becomes extinct and cannot be used after once use.

Particularly, because the screening process is performed sequentially,it takes a few days to a few weeks to obtain a result though there issomewhat a difference depending on target materials.

As described above, the conventional art has a drawback in that it takesa long time and makes many efforts to search new materials using PS-SPCLand it continuously consumes the high-priced peptide library in view ofthe characteristic of the PS-SPCL.

DISCLOSURE OF INVENTION Technical Problem

The present invention has been made to solve the foregoing problems withthe prior art, and therefore the present invention provides a PS-SPCLsearching apparatus and method using SPR, for displaying the result ofPS-SPCL searching as an image using SPR, thereby greatly reducing asearching time and efforts made.

Technical Solution

According to an aspect of the present invention, a PositionalScanning—Synthetic Peptide Combinatorial Library (PS-SPCL) searchingmethod using Surface Plasmon Resonance (SPR) is provided. The methodincludes spotting and fixing each of a plurality of peptide pools to atop of one thin metal film; inputting specific materials to the top ofthe thin metal film; applying a Transverse Magnetic (TM) mode light to abottom of the thin metal film and exciting SPR for the thin metal film;and detecting a TM mode reflected light reflected from the thin metalfilm and displaying the detected light as a two-dimensional image.

In spotting and fixing each of the plurality of peptide pools to the topof the thin metal film, the plurality of peptide pools may be fixed tothe surface of the thin metal by a covalent bond.

The thin metal film may be a metal emitting electron by an externalstimulus and having a negative dielectric constant.

The method may further include, before spotting and fixing each of theplurality of peptide pools to the top of the thin metal film,synthesizing the plurality of peptide pools so that peptides come incontact with the surface of the thin metal film while forming a covalentbond by manipulating N-terminals or C-terminals of the peptides.

The method may further include detecting optimal amino acids by positionfrom the two-dimensional image.

According to another aspect of the present invention, a PositionalScanning—Synthetic Peptide Combinatorial Library (PS-SPC) searchingapparatus using Surface Plasmon Resonance (SPR) is provided. Theapparatus includes a PS-SPCL chip including a thin metal film and a flattype transparent dielectric substrate for supporting the thin metalfilm, an incident light provision unit, a prism, and a reflected lightdetector. The thin metal film spots and fixes one or more peptide pools.The flat type transparent dielectric substrate supports the thin metalfilm. The incident light provision unit provides a TM mode incidentlight for exciting SPR. The prism propagates the incident light, whichis provided by the incident light provision unit, from a bottom of thePS-SPCL chip to an interface between the thin metal film and the flattype transparent dielectric substrate and emits a reflected lightreflected from the thin metal film. The reflected light detector detectsa reflected light emitted through the prism and displays the detectedlight as a two-dimensional image.

The thin metal film may be a metal emitting electron by an externalstimulus and having a negative dielectric constant.

The flat type transparent dielectric substrate may be formed of highrefractive-index polymer materials comprising slide glass or CycloOlefinCopolymer (COC).

The apparatus may further include a signal processor for analyzing thetwo-dimensional image displayed by the reflected light detector anddetecting optimal amino acids by position as amino acids that are fixedto peptide pools appearing most bright on the two-dimensional imageamong a peptide pool group whose different amino acids are fixed in thesame position.

Advantageous Effects

As set forth above, the present invention has an excellent effect ofenabling observations of the degree of each reaction between peptidepools and target materials, which induces a change of an SPR condition,at a time by fixing all of a set of peptide pools to a thin metal filmin a constant array, then making the target materials flow to the thinmetal film to induce the reaction between the peptide pools and thetarget materials, then making light incident on the thin metal film andexciting SPR, and then processing a reflected light reflected from thethin metal film into a two-dimensional image. Also, the presentinvention has an excellent effect of enabling the reuse of the thinmetal film to which a plurality of the peptides pools are fixed, byremoving only the target materials by a physiochemical method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating an example of PS-SPCL;

FIG. 2 is a diagram illustrating an example of a conventional searchingprocess using PS-SPCL;

FIG. 3 is a schematic diagram illustrating a basic construction of anSPR sensor;

FIG. 4 is a mimetic diagram illustrating a PS-SPCL chip according to anexemplary embodiment of the present invention;

FIG. 5 is a diagram illustrating the entire construction of a PS-SPCLsearching apparatus using SPR according to an exemplary embodiment ofthe present invention; and

FIG. 6 is a view showing an example of a two-dimensional image showingthe result of PS-SPCL searching using SPR according to an exemplaryembodiment of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

Preferred embodiments of the present invention will be described hereinbelow with reference to the accompanying drawings. In the followingdescription, well-known functions or constructions are not described indetail since they would obscure the invention in unnecessary detail.

Throughout the drawings, it should be noted that like reference numbersare used to depict the same or similar elements, features andstructures.

Throughout the specification, ‘connecting of any part with other part’includes not only ‘directly connecting’ but also ‘indirectly connecting’with another element interposed between them. ‘Including any element’signifies not excluding other element but being able to further includeother element unless otherwise indicated.

In searching new materials using PS-SPCL, the present invention is todivide a set of peptide pools of PS-SPCL on a group basis, then spot andfix the peptide pools to a surface of a thin metal film supportingsurface plasmon, and then input target materials to the surface of thethin metal film, thereby enabling observations of an interaction betweenmolecules, which are made in each peptide pool, through an SPR imagingtechnique. The present invention is to enable observations of thereaction of the set of peptide pools at a time and search for optimalreaction materials to target materials easily and simply, by makingobservations of a two-dimensional image obtained through the SPR imagingtechnique.

In order to help understanding of the present invention, a briefdescription of an SPR sensor is first made below.

FIG. 3 is a schematic diagram illustrating a basic principle of the SPRsensor.

Referring to FIG. 3, in the SPR sensor, if a Transverse Magnetic (TM)polarized light is incident on a thin metal film 22 through a prism 21,SPR takes place at specific incidence angle and thickness of the thinmetal film 22. At this time, a phenomenon in which the energy of theincident light is absorbed into the thin metal film 22 and thus, inducesa sudden decrease of the intensity of a reflected light reflected fromthe thin metal film 22 occurs.

The SPR is influenced by an incident angle and an effective thickness ofthe thin metal film 22. Therefore, target materials 24 a contained in asample 24 are coupled with fixed biomaterials 23 and thus a change of aneffective thickness is induced, provided that with the incident anglefixed, the biomaterials 23 peculiarly coupled with the target materials24 a are fixed to a surface of the thin metal film 22 and the sample 24is inputted to the surface of the thin metal film 22. Accordingly, byanalyzing a reflected light reflected from the thin metal film 22, theexistence or absence of the target materials 24 a and the concentrationof the target materials 24 a can be quantitatively detected. Further, incases where a reflected light from the thin metal film 22 is displayedas a two-dimensional image using a Charge-Coupled Device (CCD), visualobservations can be made of a bio reaction at a surface of the thinmetal film 22 by virtue of the fact that a portion of thetwo-dimensional image where the intensity of the reflected light is highis displayed bright and a portion of the two-dimensional image where theintensity of the reflected light is low is displayed dark.

A method for searching new materials by PS-SPCL using the principle ofSPR is described with reference to FIGS. 4 to 6 below.

FIG. 4 is a perspective diagram illustrating a PS-SPCL chip in whichpeptide pools of PS-SPCL are spotted and fixed according to an exemplaryembodiment of the present invention.

As shown in FIG. 4, a plurality of peptide pools 33 included in a set ofPS-SPCLs are spotted and fixed at predetermined intervals onto a thinmetal film 32 formed on a flat type transparent dielectric substrate 31according to an exemplary embodiment of the present invention. A PS-SPCLchip is the inclusion of the substrate 31 and the thin metal film 32 towhich the peptide pools 33 are fixed.

The thin metal film 32 is a metal which supports a surface plasmonphenomenon formed at a predetermined thickness (e.g., a fewmicrometers). The thin metal film 32 may be formed of metal such asaurum (Au), argentums (Ag) copper (Cu), and aluminum (Al) that emitelectron by an external stimulus and have negative dielectric constants.In general, the thin metal film 32 uses argentums (Ag) showing thesharpest SPR peak and aurums (Au) having superior surface stability.

The flat type transparent dielectric substrate 31 serves to support thethin metal film 32. The transparent dielectric substrate 31 is formed oftransparent materials to propagate an incident light incident through aprism for exciting SPR to the thin metal film 32. In general, thetransparent dielectric substrate 31 can be formed of transparent plasticformed of slide glass and high refractive-index polymer. Also, thetransparent dielectric substrate 31 can be formed of polymer materialssuch as CycloOlefin Copolymer (COC).

In general, a set of PS-SPCLs consists of a hundred peptide pools. Inthe peptide pools each, specific amino acids are fixed in specificpositions and random amino acids are arrayed in remaining positions.Here, fixed amino acids or fixing positions are different at eachpeptide pool. That is, as mentioned in FIG. 1, the peptide pools aregrouped into a few groups and fixing positions or fixed specific aminoacids are the same by group. In peptide pools of the same group,different specific amino acids are fixed in the same position or thesame specific amino acids are fixed in different positions.

In the present invention, the peptide pools of a set are spotted atpredetermined intervals on the thin metal film 32. In a peptide poolsynthesis process, peptides come in contact with the surface of the thinmetal film 32 while forming a covalent bond by manipulating N-terminalsor C-terminals of the peptides.

For example, in a PS-SPCL synthesis process, the functional groupconsisting of a hydrogen atom (-SH) is added to the N-terminals orC-terminals and then PS-SPCL is spotted to the thin metal film 32 usinggeneral equipment such as a DeoxyriboNucleic Acid (DNA) arrayer. At thistime, a surface of the thin metal film 32 reacts with peptides of thespotted PS-SPCL because the surface of the thin metal film 32 has beenactivated. As a result, the peptides are fixed to the surface of thethin metal film 32 by a covalent bond.

In this exemplary embodiment, a fixing method using a covalent bond isexemplified but this does not intend to limit a method of fixing thepeptide pools to the thin metal film 32 in the present invention. Thepresent invention can adopt one of several chemical methods well knownto the public.

The fixed PS-SPCL can vary in length from trimer to octamer according toneed. Accordingly, spotted peptide pools can be different in number andare not limited particularly in length and number.

Among constituent amino acids of PS-SPCL used for fixing, specific aminoacids (e.g., cysteine) can be excluded according to application example.However, this is merely an example but does not intend to limitconstituent amino acids of PS-SPCL used in the present invention.

If the peptide pools of the PS-SPCLs are fixed onto the thin metal film32 as described above, the PS-SPCL chip is installed in an SPR sensor,then specific materials that are targets for analysis are inputted, andthen a reflected light obtained is converted into a two-dimensionalimage by enabling the SPR sensor.

FIG. 5 is a diagram illustrating a state where the PS-SPCL chip ismounted in an SPR sensor to perform PS-SPCL searching using SPRaccording to an exemplary embodiment of the present invention. That is,FIG. 5 shows the entire scheme of a PS-SPCL searching apparatus usingSPR according to an exemplary embodiment of the present invention.

Referring to FIG. 5, the PS-SPCL searching apparatus can include aPS-SPCL chip, an incident light provision unit 45, a prism 41, areflected light detector 46, and a signal processor 47. The PS-SPCL chipincludes a thin metal film 32 and a flat type transparent dielectricsubstrate 31. The thin metal film 32 spots and fixes one or more peptidepools 33 of PS-SPCL. The flat type transparent dielectric substrate 31supports the thin metal film 32. The incident light provision unit 45provides a TM mode incident light for exciting SPR. The prism 41propagates an incident light provided from the incident light provisionunit 45 to an interface between the thin metal film 32 and the flat typetransparent dielectric substrate 31 and emits a reflected lightreflected from the thin metal film 32. The reflected light detector 46detects a reflected light emitted through the prism 41 and displays theemitted light as a two-dimensional image.

In more detail, on the prism 41 of the SPR image sensor is disposed thePS-SPCL chip that includes the thin metal film 32 to which the peptidepools 33 each are fixed and the flat type transparent dielectricsubstrate 31.

Then, target materials 42 of a liquefied state flow on the thin metalfilm 32 to which the peptide pools 33 of the PS-SPCL are fixed, toinduce an interaction between the target materials 42 and the peptidepools 33. Then, a TM polarized incident light 43 is irradiated into theprism 41. Then, a reflected light 44 is emitted from the prism 41 and isdetected and processed into a two-dimensional image.

The incident light 43 can be provided from the incident light provisionunit 45. The incident light provision unit 45 includes a light sourceand a polarizer for TM polarizing light generated from the light source.The two-dimensional image can be realized using the reflected lightdetector 46. The reflected light detector 46 includes a two-dimensionallight receiving unit (e.g., a Charge-Coupled Device (CCD) or aComplementary Metal Oxide Semiconductor (CMOS) imaging sensor) and aprojection screen or a device for detecting the brightness of each pointon a two-dimensional plane.

The TM mode incident light incident on the prism 41 enters the thinmetal film 32, thereby exciting SPR on the thin metal film 32.

Because the peptide pools 33 of the PS-SPCL are fixed to the surface ofthe thin metal film 32, an SPR condition is different in each portion ofthe thin metal film 32 where the peptide pools 33 are spotted dependingon the degree of reaction between the fixed peptide pools 33 and thetarget materials 42.

In more detail, in cases where light is incident at an SPR angle, at aportion of the thin metal film 32 where coupling with the targetmaterials 42 much occurs, an effective thickness of a surface increasesand is far beyond the SPR condition and thus, the intensity of areflected light increases. On the other hand, at a portion of the thinmetal film 32 where coupling with the target materials 42 less occurs,SPR takes place and the energy of the incident light is much propagatedto the thin metal film 32 and thus, the intensity of a reflected lightdecreases.

That is, the intensity of a reflected light reflected from the thinmetal film 32 is in inverse proportional to the degree of couplingbetween the target materials 42 and the peptide pools 33.

Thus, if the reflected light is processed into a two-dimensional image,the reaction of a plurality of the peptide pools 33 can be identified ata time through the fact that a difference of light and darkness takesplace in each peptide pool because the intensity of the reflected lightis different depending on the degree of coupling of the peptide pools33. As a result, it can be easily identified that which are optimalamino acids by position.

FIG. 6 is a view showing an example of a two-dimensional image showingthe searching result of PS-SPCL searching using SPR according to anexemplary embodiment of the present invention. Here, a vertical axisdenotes positions where a specific amino acid is fixed at each row and ahorizontal axis denotes a specific amino acid fixed at each column.

Referring to FIG. 6, a different specific amino acid of a peptide poolgroup of a first row is fixed in a first position of a sequence, adifferent specific amino acid of a peptide pool group of a second row isfixed in a second position of a sequence, and a different specific aminoacid of a peptide pool group of a third row is fixed in a third positionof a sequence. Also, it can be noted that an amino acid ‘A’ is fixedamong a peptide pool group of a first column and an amino acid ‘D’ isfixed among a peptide pool group of a second column.

In the two-dimensional image, the brighter means the larger a couplingforce with specific materials. That is, in the two-dimensional image ofthe reflected light of FIG. 6, it can be noted that ‘E’ (glutamate) of afirst position of a sequence is an optimal amino acid and ‘F’(phenylalanine) of a second position of a sequence is an optimal aminoacid.

As such, the present invention can obtain information on an optimalamino acid of each position through a single searching work only.

For this, the PS-SPCL searching apparatus of the present invention canfurther include the signal processor 47 for analyzing a two-dimensionalimage provided from the reflected light detector 46 and detecting anoptimal amino acid in each position.

After searching is completed, the PS-SPCL chip can be reused by removingthe target materials 42 inputted to the thin metal film 32 by virtue ofa chaotropic reagent such as urea, guanidine hydrochloride or adetergent such as sodium dodecyl sulfate. In more detail, the PS-SPCLchip can be reused through simple physiochemical washing mentioned abovebecause the peptide pools 33 are fixed to the surface of the thin metalfilm 32 by a covalent bond. The above washing does not certainly intendto limit a physiochemical method for the reuse of the PS-SPCL chip.

While the present invention has been shown and described in connectionwith the exemplary embodiments, it will be apparent to those skilled inthe art that modifications and variations can be made without departingfrom the spirit and scope of the invention as defined by the appendedclaims.

1. A Positional Scanning—Synthetic Peptide Combinatorial Library(PS-SPCL) searching method using Surface Plasmon Resonance (SPR), themethod comprising: spotting and fixing each of a plurality of peptidepools to a top of one thin metal film; inputting specific materials tothe top of the thin metal film; applying a Transverse Magnetic (TM) modelight to a bottom of the thin metal film and exciting SPR for the thinmetal film; and detecting a TM mode reflected light reflected from thethin metal film and displaying the detected light as a two-dimensionalimage.
 2. The method of claim 1, wherein in spotting and fixing each ofthe plurality of peptide pools to the top of the thin metal film, theplurality of peptide pools are fixed to the surface of the thin metal bya covalent bond.
 3. The method of claim 1, wherein the thin metal filmis a metal emitting electron by an external stimulus and having anegative dielectric constant.
 4. The method of claim 2, furthercomprising: before spotting and fixing each of the plurality of peptidepools to the top of the thin metal film, synthesizing the plurality ofpeptide pools so that peptides come in contact with the surface of thethin metal film while forming a covalent bond by manipulatingN-terminals or C-terminals of the peptides.
 5. The method of claim 4,wherein in synthesizing the plurality of peptide pools, the functionalgroup consisting of a hydrogen atom (—SH) is added to the N-terminals orC-terminals.
 6. The method of claim 4, wherein in detecting the TM-modereflected light reflected from the thin metal film and displaying thedetected light as the two-dimensional image, brightness of each point ona two-dimensional plane is detected through a Charge-Coupled Device(CCD) or a Complementary Metal Oxide Semiconductor (CMOS) image sensor.7. The method of claim 1, further comprising: detecting optimal aminoacids by position from the two-dimensional image.
 8. The method of claim7, wherein in detecting the optimal amino acids by position, the optimalamino acids of the position are detected as amino acids that are fixedto peptide pools appearing most bright on the two-dimensional imageamong a peptide pool group whose different amino acids are fixed in thesame position.
 9. The method of claim 1, further comprising: afterdisplaying as the two-dimensional image, removing the target materialsinputted to the top of the thin metal film by a physical or chemicalmethod.
 10. The method of claim 9, wherein in removing the targetmaterials by the physical or chemical method, the target materials areremoved using a chaotropic reagent such as urea, guanidine hydrochlorideor a detergent such as sodium dodecyl sulfate.
 11. A PositionalScanning—Synthetic Peptide Combinatorial Library (PS-SPCL) searchingapparatus using Surface Plasmon Resonance (SPR), the apparatuscomprising: a PS-SPCL chip comprising: a thin metal film for spottingand fixing one or more peptide pools; and a flat type transparentdielectric substrate for supporting the thin metal film; an incidentlight provision unit for providing a TM mode incident light for excitingSPR; a prism for propagating the incident light, which is provided bythe incident light provision unit, from a bottom of the PS-SPCL chip toan interface between the thin metal film and the flat type transparentdielectric substrate and emitting a reflected light reflected from thethin metal film; and a reflected light detector for detecting areflected light emitted through the prism and displaying the detectedlight as a two-dimensional image.
 12. The apparatus of claim 11, whereinthe thin metal film is a metal emitting electron by an external stimulusand having a negative dielectric constant.
 13. The apparatus of claim11, wherein the flat type transparent dielectric substrate is formed ofhigh refractive-index polymer materials comprising slide glass orCycloOlefin Copolymer (COC).
 14. The apparatus of claim 11, wherein theplurality of peptide pools are coupled by a covalent bond with a surfaceof the thin metal film by manipulating N-terminals or C-terminals. 15.The apparatus of claim 11, wherein the reflected light detectorcomprises a Charge-Coupled Device (CCD) or a Complementary Metal OxideSemi-conductor (CMOS) image sensor.
 16. The apparatus of claim 11,further comprising: a signal processor for analyzing the two-dimensionalimage displayed by the reflected light detector and detecting optimalamino acids by position as amino acids that are fixed to peptide poolsappearing most bright on the two-dimensional image among a peptide poolgroup whose different amino acids are fixed in the same position.